The intoxication of eukaryotic cells by microbial and plant toxins involves the following steps: (i) binding of these proteins to their respective cell-surface receptors, (ii) receptor-medicated endocytosis of the receptor/toxin complex into endocytic vesicles, and (iii) the facilitated delivery of their respective catalytic domains to the eukaryotic cell cytosol. Of all these processes, the fundamental mechanisms involved in the delivery of the catalytic domain to the cytosol is the least well understood. The specific aims of this proposal are focused on the analysis of transmembrane domain and catalytic domain mutants which are defective in the translocation of the catalytic domain to the cytosol of target cells. We propose the construction and analysis of epitope tagged mutants in the fusion toxin DAB389IL-2 to gain further insight into the mechanism of translocation. The recent development of an in vitro catalytic domain translocation assay using purified endocytic vesicles offers a powerful new assay in the study of this process. Since this system requires ATP and cytosolic factors, it is likely that we should uncover toxin/target cell protein interactions that were hither to unknown. DAB389-IL2 is extremely potent (e.g., IC50 < 5 x 10-12 M for target cells which carry the high affinity IL-2 receptor) and the difference between receptor-mediated and non-specific toxicity is > 5-logs of fusion toxin concentration. In addition, the level of non-specific binding of this system has allowed the detection of subtle changes in the interaction between the fusion protein and the target cell. Since DAB389-IL2 and diphtheria toxin appear to follow an identical route of entry into the cell, we believe that an increased understanding of the facilitated delivery of the catalytic domain will be broadly applicable to the delivery of other microbial and plant toxins.